hPSCs and lt-NES cells can be transfected with unmodified mRNA

To confirm that hPSCs can be transfected with unmodified mRNA, hiPSCs of the line iLB-30-r12 and hESCs of the H9.2 and I3 lines were plated on 6-well dishes. 24 hours later, they were transfected with 800 ng/well of modified mRNA or 800 ng of unmodified mRNA. As a control, cells were transfected with the same amount of polyA, a polynucleotide. The transfection reagent used was Lipofectamine® LTX. 16

hours after transfection, the transfection mix was exchanged with fresh medium and pictures have been taken to estimate the fluorescence. To facilitate comparison of the fluorescence intensities and efficiency of the transfection, the fluorescence pictures were taken using the same exposure time for each experiment.

Figure 3.15: Transfection of unmodified GFP mRNA into hiPSCs results in a robust translation of GFP

Transfection of iLB-30-r12 iPSCs with 800 ng of unmodified mRNA leads to a stronger fluorescence signal than with modified mRNA.

Magnification: 40X; exposure time= 125ms

The human iPSC line iLB-30-r12 could be transfected efficiently with modified and unmodified synthetic GFP mRNA. As it can be seen in Figure 3.15, both cells transfected with modified and unmodified mRNA survived mRNA transfection. The colonies have a similar size and a similar amount of debris is present in the supernatant, indicating no additional stress induced by unmodified synthetic mRNA.

Transfection with unmodified mRNA seems to lead to stronger fluorescence intensity, which could be due to a better translation. Both fluorescence pictures depicted in Figure 3.15 were exposed for 125ms.

A similar effect could be observed in H9.2 human embryonic stem cells (Figure 3.16);

however, the effect was less striking as the difference between the fluorescence intensities of cells transfected with modified and unmodified GFP mRNA was not as strong as observed in iLB-30-r12 hiPSCs. Nonetheless, the transfection efficiency of

Figure 3.16: Transfection of both modified and unmodified GFP mRNA leads to an intense translation of GFP

The intensities of the fluorescence are on a comparable level for modified and unmodified GFP mRNA transfected into hESCs of the H9.2 line. Both mRNA can be transfected efficiently into the hESCs.

Magnification: 40X; exposure time= 100ms

With I3, another human embryonic stem cell line was tested. Transfection with either modified or unmodified mRNA (Figure 3.17) as well as polyA control (data not shown) resulted in decreased growing or increased dying of the cells as only little cells were attached after 16 hours of incubation with the transfection mix. In spite of this, the surviving cells were transfected with a high efficiency. The synthetic mRNA appeared to be translated very efficiently, as an exposure time of 75ms was sufficient to detect fluorescence in the modified mRNA transfected cells as well as the unmodified, which was even stronger (Figure 3.17).

Figure 3.17: hESCs translate both modified and unmodified synthetic GFP mRNA The hESC line I3 shows a similar transfection capability as iLB-30-r12 hiPSCs. The intensity of the fluorescence is stronger in case of unmodified mRNA transfection. Both mRNAs can be transfected very efficiently into the hESCs.

Magnification: 40X; exposure time= 75ms

In order to quantify the observed effects, flow cytometric analyses were performed.

For that, the hiPSC line iLB-30-r12 and the hESC lines H9.2 and I3 were transfected as described before. For flow cytometry, cells had to be singularized.

In Figure 3.18 exemplary histograms of the flow cytometry are shown. iLB-30-r12 cells could be transfected with modified GFP mRNA with an efficiency of 51.9%.

When transfected with unmodified mRNA, efficiency increased up to 64.5%. Notably, in the overlay the stronger intensity of the unmodified mRNA (blue line) compared to the modified mRNA (red line) can be seen as the blue curve shifts to the right compared to the red one.

For H9.2, this is not true, which recapitulates the results seen in Figure 3.16. The intensities of both mRNA seem to be comparable, as both lines have the peak at the same point. Nevertheless, the efficiency of transfection with unmodified mRNA was higher (56.2%) than with modified mRNA (40.6%).

The findings found for I3 hESCs via microscopy could be confirmed by flow cytometry as well (Figure 3.18). The blue line has its peak at the far right side of the histogram, indicating a stronger fluorescence than the cells transfected with modified mRNA, as its red line shows a peak more to the left. The efficiencies of transfection

were high, as it was expected after microscopic evaluation (Figure 3.17); transfection with modified mRNA resulted in an efficiency of 67.7%, the use of unmodified mRNA could increase the efficiency up to 78%.

Figure 3.18: Increased efficiency and stronger GFP intensity after transfection with unmodified GFP mRNA in hPSCs

Exemplary histograms show that the transfection efficiency rises from 51.9% to 64.5% (iLB-30-r12), from 40.6% to 56.2% (H9.2) and from 67.7% to 78% (I3) when hPSCs are transfected with unmodified mRNA instead of modified mRNA. In the overlays, the blue line shows that the intensity is stronger in cells transfected with unmodified mRNA compared to modified mRNA (red line)

Blue: unmodified; red: modified

The results shown in Figure 3.18 are just exemplarily, as the histograms are shown only from single experiments. In total, hPSCs were transfected in seven individual experiments with modified and unmodified mRNA.

Figure 3.19: Transfection of hPSCs with unmodified mRNA leads to a significantly higher efficiency

Seven individual transfection attempts result in a mean efficiency for transfection with modified mRNA of 41.83% (±18.44). Efficiency is increased up to 51.57% (±18.23%) for unmodified mRNA.

n=7; *: p<0.05 according to a paired t-test

These experiments resulted in an average transfection efficiency for modified mRNA of 41.83% (Figure 3.19). Transfection with unmodified GFP mRNA could be achieved with an average efficiency of 51.57%. The standard deviation of 18% in both cases was relatively high, but still a t-test resulted in a p-value lower than 0.05, as a paired t-test was performed. The variety between the efficiencies in the seven experiments was high, but the increase of the efficiencies when unmodified mRNA was transfected did always occur.

Taken together, these results let suggest that hPSCs cannot only be transfected with unmodified synthetic mRNA, but that the efficiency is even increased when unmodified instead of modified mRNA is transfected. If this were due to a reduced innate immune system response, it would be interesting to investigate how derivatives of hPSCs react upon transfection with unmodified synthetic mRNA.

In order to elucidate the immune status of hPSC-derivatives, lt-NES cells, which were differentiated from the iPSC line iLB-30-r12, were transfected with modified and unmodified GFP mRNA. Per well of a 6-well dish, 800 ng of the designated mRNA were transfected into lt-NES with Lipofectamine® LTX. As a control, 800 ng of the polyA polynucleotide were transfected (data not shown).

16 hours after transfection, efficiency and intensity were approximated via fluorescence microscopy.

Figure 3.20: Transfection of unmodified GFP mRNA into lt-NES cells results in a robust translation of GFP

Transfection of unmodified GFP mRNA shows higher fluorescence intensity. Efficiency appears to be higher for unmodified mRNA.

Magnification: 40X; exposure time= 75ms

lt-NES cells showed the same reaction as hPSCs upon transfection with unmodified GFP mRNA (Figure 3.20). Apparently, the fluorescence intensity of the cells transfected with unmodified mRNA once more was stronger. Efficiency seemed to be increased as well. The cells kept growing in the same instance, no increased cell death indicated by detached cells in the supernatant could be observed.

Quantification with flow cytometry confirmed that the efficiency as well as the intensity increased when unmodified GFP mRNA was transfected (Figure 3.21). Flow cytometry revealed an efficiency of 23.1% for the modified mRNA, whereas the unmodified mRNA could be transfected with an efficiency of 38.3% into lt-NES cells.

In the overlay, the blue curve, which represents the histogram for the cells transfected with unmodified mRNA, is shifted to the right, indicating a strong fluorescence intensity.

Figure 3.21: Increased efficiency and stronger GFP intensity in lt-NES cells after transfection with unmodified GFP mRNA

lt-NES cells can be transfected more efficiently with unmodified GFP mRNA (38.3%) than with modified GFP mRNA (23.1%). The fluorescence intensity is stronger when lt-NES cells are transfected with unmodified mRNA as in the overlay the peak of the blue histogram (unmodified) is more to the right than the peak of the histogram of modified mRNA-transfected cells (red).

Transfection of lt-NES cells was carried out three times. The variety between the individual experiments was relatively high as indicated by the high standard deviation (Figure 3.22). Nonetheless, in each experiment the efficiency was higher when unmodified mRNA was transfected.

Figure 3.22: Transfection of lt-NES cells with unmodified mRNA leads to an increased efficiency

The mean transfection efficiency increases from 28.87% (±17.91%) for modified mRNA up to 43.63% (±23.57%) for unmodified mRNA.

n=3; error bars show standard deviation; n.s.: not significant; p= 0.06 according to a paired t-test

The average efficiency for modified mRNA was 28.87%. It increased up to 48.63%

for unmodified mRNA. Due to a p-value of 0.06, the increase of the efficiency upon transfection with unmodified mRNA can only be considered as a trend, as it is not statistically significant.